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Improvement of probe peptides for coiled‐coil labeling by introducing phosphoserines
Author(s) -
Ono Satoshi,
Yano Yoshiaki,
Matsuzaki Katsumi
Publication year - 2012
Publication title -
peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.22059
Subject(s) - chemistry , hek 293 cells , fluorophore , membrane , hela , cell , microbiology and biotechnology , biophysics , fluorescence , biochemistry , gene , biology , physics , quantum mechanics
We have developed a method of rapidly labeling membrane proteins in living cells using a high‐affinity heterodimeric coiled‐coil construct containing an E3 tag (EIAALEK) 3 genetically fused to the target protein and a K4 probe (KIAALKE) 4 labeled with a fluorophore such as tetramethylrhodamine (TMR) at its N‐terminus (TMR‐K4). However, coiled‐coil labeling cannot be applied to highly negatively charged cell lines such as HEK293, because of the nonspecific adsorption of the positively charged K4 probes to cell membranes. To reduce the net positive charge, we synthesized new probes that include phosphoserine residues (pSer) between the K4 sequence and TMR fluorophore (TMR‐(pSer) n ‐K4, [n = 1–3]). The affinity of the pSer‐introduced probes was comparable to that of the TMR‐K4 probe. However, the TMR‐(pSer) 2 ‐K4 and TMR‐(pSer) 3 ‐K4 probes tended to aggregate during labeling. In contrast, TMR‐pSer‐K4, which was as soluble as TMR‐K4, achieved higher signal/background ratios (30–100) for four host cell lines (HEK293, HeLa, SH‐SY5Y, and PC12) than did TMR‐K4 (∼10 for HEK293 cells), demonstrating that the improved probe can be used for various types of cells. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 234–238, 2012.